The DNA replication system of bacteriophage T4 is a relatively simple system, yet it has functional homology to the replication system organisms such as E. coli, yeast, and humans. This proposal seeks to investigate the structure and mechanism of assembly of the T4 holoenzyme, which consists of the DNA polymerase, the sliding clamp, and the clamp loader. The points of interaction between the polymerase and the sliding clamp will be investigated by photo crosslinking followed by proteolysis and sequencing. Previous studies as well as preliminary results indicate that the interaction point of the polymerase is at its C-terminus. The polymerase will therefore be derivatized with a photo cross linker at its C-terminus using a self-cleaving protein. The mechanism of loading the sliding clamp onto DNA, which involves opening the circular clamp trimer and concatenating it onto the DNA, will be investigated using a sliding clamp mutant with a disulfide bond that imposes a topological constraint. Preliminary studies demonstrated that this topological constrain does indeed inhibit the loading of the sliding clamp onto DNA. Fluorescence studies will further characterize the mechanistic point of inhibition. Finally, the clamp loader will be tested to determine whether it acts a prolyl isomerase in loading the sliding clamp onto DNA. These experiments will provide additional insight into the molecular interactions in the T4 holoenzyme.